Joyce C M, Grindley N D
Proc Natl Acad Sci U S A. 1983 Apr;80(7):1830-4. doi: 10.1073/pnas.80.7.1830.
Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems.
利用现有的基因融合技术，我们构建了一些质粒，这些质粒可指导DNA聚合酶I羧基末端三分之二的过量表达，这部分对应于通过蛋白水解获得的“克列诺片段”。我们使用了两种构建体，一种是由lac启动子驱动表达，另一种是由噬菌体λ的左向启动子驱动表达，均获得了占细胞蛋白百分之几的过量表达量。通过一个快速的三步纯化程序，从这些过量表达菌株中已将聚合酶片段纯化至同质状态，得到的物质能够进行与蛋白水解获得的片段相同的反应（聚合反应、3'标记、DNA序列分析）。此类过量表达菌株的获得应极大地促进对DNA聚合酶I的结构和机制研究。此外，我们所描述的用于克隆和表达基因片段的技术应普遍适用于其他系统中蛋白质结构和功能的研究。
